Project Summary Esophageal squamous epithelium comprises a basal layer of proliferative cells that undergoes differentiation in the suprabasal layer and luminal desquamation, facilitating epithelial renewal. Although stem cells responsible for and Through work supported by my K01 grant, we have recently demonstrated that autophagy-mediated modulation of oxidative stress and mitochondrial function supports expansion of cancer stem cells in the tumor microenvironment; however, what role, if any, autophagy plays in normal esophageal stem cell biology remains to be determined. Our published and preliminary data demonstrate evidence of autophagy in a subset of basal cells in murine and human esophageal epithelium in situ. Additionally, pharmacologic or genetic autophagy impairment enhances 3D esophageal organoid formation ex vivo. The goal of the current R03 proposal is to investigate the role of autophagy in regulating basal cell dynamics, namely the balance between actively proliferating and quiescent stem cells. We hypothesize that autophagy limits basal cell proliferation to maintain a pool of slow-cycling esophageal stem/progenitor cells. To test this hypothesis we will pursue the following Specific Aims: Aim 1: To determine the relation relationship between autophagy level and the basal cell stemness/proliferation axis in esophageal epithelium. Aim 2: To define the functional consequences of genetic autophagy impairment upon esophageal basal cell dynamics. These innovative studies utilize functional evaluation of human endoscopic tissue biopsies, 3D esophageal organoids, RNA-sequencing, and a genetically engineered murine model with lineage tracing capacity to investigate autophagy as a novel regulator of esophageal basal cell dynamics, building a platform for future investigations into the specific molecular mechanisms underlying autophagy-mediated esophageal cell fate determination under conditions of health and disease. This R03 proposal represents a logical progression from my K01 grant seeking to establish mitophagy as a critical mediator of epithelial-mesenchymal transition under conditions of homeostasis and carcinogenesis and will facilitate expansion of my research program into the exciting field of stem cell biology. These studies will lay the foundation for an NIH R01 proposal, thereby supporting my transition to a fully independent investigator. the maintenance of esophageal epithelium characterization of these cells remains are thought to reside in the basal cell compartment, identification elusive.